Onchocercal Keratitis Research Articles

Angiogenic activity of Onchocerca volvulus recombinant proteins similar to vespid venom antigen 5

Although the mechanisms underlying the host inflammatory response in ocular onchocerciasis have been examined, the role of particular parasite proteins in this process remains largely unexplored. Recently, it was found that one of the most abundant expressed sequence tags in Onchocerca volvulus infective larvae encoded a protein with similarities to a component of vespid venom. This clone was designated O. volvulus Activation associated Secreted Protein -1 ( Ov-asp-1). We report the characterization of three members of a family of proteins, designated the Ov-ASP family, of which Ov-ASP-1 is a member. Sequence based and phylogenetic analyses suggest that these proteins form a filarial specific protein family related to both the vespid venom antigen 5 and the vertebrate CRISP/Tpx family of proteins. The three members of the Ov-ASP family exhibit distinct patterns of expression in the life cycle of O. volvulus. Genomic Southern blot analyses indicate that several genes encoding sequences related to the Ov- asp family are present in the genome of O. volvulus. Recombinant proteins expressed from full length cDNAs encoding two members of the Ov- asp family were found to induce an angiogenic response after injection into corneas of naive mice, and vessel formation was associated with only minor inflammatory cell infiltration. These data suggest that Ov-ASP proteins may directly induce an angiogenic response and may therefore contribute to corneal neovascularization in onchocercal keratitis.

IL-12 exacerbates helminth-mediated corneal pathology by augmenting inflammatory cell recruitment and chemokine expression.

Corneal inflammation (keratitis) is a major cause of visual impairment in Onchocerca volvulus infection. Previous studies showed that onchocercal keratitis can be induced in mice following s.c. immunization and intracorneal injection with soluble O. volvulus Ags (OvAg), and that the inflammatory response is dependent on T cells and IL-4. Since recombinant IL-12 impairs IL-4-dependent, Th2-mediated responses in other parasitic infections and in models of allergic asthma, the present study was undertaken to determine the effect of IL-12 on onchocercal keratitis. Mice were injected i.p. with IL-12 or saline at the time of initial sensitization to OvAg. Surprisingly, IL-12 treatment caused significant exacerbation of corneal pathology, which was associated with increased eosinophil and mononuclear cell infiltration into the corneal stroma. Consistent with the well-documented effect of IL-12 on Th1 cell development, corneas of IL-12-treated animals had elevated expression of the Th1 cytokine IFN-gamma and diminished expression of the Th2 cytokines IL-4, IL-5, IL-10, and IL-13. However, corneas from these animals also had marked elevation of alpha- and beta-chemokines known to be active on eosinophils and mononuclear cells, including IFN-gamma-inducible protein (IP)-10, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, JE/monocyte chemotactic protein-1, RANTES (regulated upon activation, normal T expressed and secreted), and eotaxin. Together, these data indicate that IL-12 exacerbates OvAg-mediated corneal pathology by enhancing chemokine expression and recruitment of inflammatory cells.

Immunopathology of onchocerciasis: a role for eosinophils in onchocercal dermatitis and keratitis.

This review has sought to demonstrate that eosinophils have an important role in the immunopathology of onchocercal dermatitis and keratitis. The most compelling evidence is the consistent presence of eosinophils and eosinophil granule proteins at the site of tissue damage, either after parasite death or direct injection of parasite antigens. A more definitive role for eosinophils in onchocercal keratitis will be determined using IL-5 gene knockout mice (an animal model of onchocercal dermatitis has yet to be established). Identification of chemoattractants and adhesion molecules necessary for eosinophil recruitment will indicate possible approaches to immune intervention.

Pleomorphism of stromal eosinophils in murine experimental onchocercal keratitis

Onchocercal keratitis (river blindness) is one of the leading worldwide causes of blindness. Light microscopic analysis of human speci-mens and corneal tissue from experimental models has implicated the eosin-ophil as an important cell in the inflammatory response. Our previous stud-ies in experimental murine onchocercal keratitis have demonstrated that the inflammatory infiltrate is composed primarily of eosinophils displaying ring shaped or bilobed nuclei. However, a number of cells were not character-izable by light microscopy, presumably due to mechanical distortion. To more fully characterize the inflammatory cell infiltrate, we examined corneal specimens by transmission electron microscopy. In addition to typical eosinophils with bilobed and ring shaped nuclei, this approach revealed cells with variable nuclear morphology and cell shape which contained the dense cored granules characteristic of eosinophils. Hence, the degree of pleomor-phsm of eosinophils is broader than appreciated and underscores the impor-tance of this cell in experimental murine onchocercal keratitis.

Onchocerca volvulus-Mediated Keratitis: Cytokine Production by IL-4-Deficient Mice

Corneal inflammation similar to human onchocercal keratitis can be induced in mice by subcutaneous immunization of a soluble extract ofOnchocerca volvulus(OvAg) followed by direct injection of OvAg into the corneal stroma. Previous studies have shown that corneal pathology is associated with increased systemic and corneal Th2 cytokine expression and that IL-4 gene knockout (IL-4−/−) mice develop less severe or noO. volvulus-mediated keratitis. The current study examined the contribution of Th2 cytokines to the diminished OvAg-induced corneal immunopathology observed in IL-4−/−mice. IL-4−/−mice (129Sv × C57B1/6), wild-type F2 littermates (IL-4+/+), and C57B1/6 mice were sensitized by repeated subcutaneous immunization with OvAg. Ten days after the final immunization, mice were sacrificed, spleens were removed, and cells were incubated with OvAg. Cells from immunocompetent C57B1/6 and IL-4+/+mice produced IL-4 and IL-5, but no IFN-γ, whereas cells from IL-4−/−mice had elevated IFN-γ but no IL-4. Interestingly, cells from these animals produced levels of IL-5 protein equivalent to those of C57B1/6 and IL-4+/+mice. To determine cytokine production in corneas during the onset of onchocercal keratitis, OvAg-immunized mice were injected intracorneally with OvAg, and cytokine gene expression in the cornea was determined by RT-PCR. Temporal analysis of cytokine gene expression in corneas of immunocompetent mice showed that the Th2-associated cytokines IL-4, IL-5, IL-10, and IL-13 were produced within 1 day of intrastromal injection, with sustained elevations for 10 days. Maximal IFN-γ mRNA levels were not detected until Day 10. This was in contrast to IL-4−/−mice in which IFN-γ appeared at Day 1 and remained elevated for at least 10 days. Moreover, in corneas from IL-4−/−mice, all Th2 cytokines with the exception of IL-4 were up-regulated and expressed with kinetics similar to that of IL-4+/+littermates. Histologically, corneas from IL-4−/−mice were less edematous and contained fewer eosinophils and other inflammatory cells than those from immunocompetent controls. As there was no difference in peripheral eosinophil levels, these data indicate that the diminished severity of onchocercal keratitis in IL-4−/−mice is not due to failure to develop systemic or local Th2 cytokine responses or to produce eosinophils, but that IL-4 may be involved in recruitment of eosinophils and other inflammatory cells into the corneal stroma.

Interleukin 4 and T helper type 2 cells are required for development of experimental onchocercal keratitis (river blindness).

Inflammation of the corneal stroma (stromal keratitis) is a serious complication of infection with the nematode parasite Onchocerca volvulus. Because stromal keratitis is believed to be immunologically mediated in humans, we used a murine model to examine the role of T cells and T helper cell cytokines in the immunopathogenesis of these eye lesions. BALB/c mice immunized subcutaneously and injected intrastromally with soluble O. volvulus antigens (OvAg) developed pronounced corneal opacification and neovascularization. The corneal stroma was edematous and contained numerous eosinophils and mononuclear cells. Stromal keratitis in immunized mice was determined to be T cell dependent based on the following observations: (a) T cell-deficient nude mice immunized and injected intrastromally with OvAg fail to develop corneal pathology; and (b) adoptive transfer of spleen cells from OvAg-immunized BALB/c mice to naive nude mice before intrastromal injection of OvAg results in development of keratitis. OvAg-stimulated lymph node and spleen cell cytokine production was dependent on CD4 cells and included interleukin (IL)-4 and IL-5, but not interferon gamma, indicating a predominant T helper type 2 cell-like response. Inflamed corneas from immunized BALB/c mice and from reconstituted nude mice had greatly elevated CD4 and IL-4 gene expression compared with interferon gamma. Mice in which the IL-4 gene was disrupted failed to develop corneal disease, demonstrating that IL-4 is essential in the immunopathogenesis of O. volvulus-mediated stromal keratitis.

Punctate keratitis induced by subconjunctivally injected microfilariae of Onchocerca lienalis.

An experimental model of human onchocercal keratitis was produced by injecting guinea pigs subconjunctivally with microfilariae (Mf) of Onchocerca lienalis. Actively motile O lienalis Mf spontaneously penetrated the central cornea and produced gray-white midstromal opacities, 0.2 to 0.4 mm in diameter, resembling those of human onchocercal punctate keratitis. Histologically, small foci of eosinophil and mononuclear cell infiltration associated with small pockets of interstitial edema were present in the central corneal stroma, with eosinophil infiltrates in the conjunctiva, episclera, limbus, and ciliary body. The severity of the punctate keratitis was increased by repeated subconjunctival inoculations of Mf. Punctate lesions were not seen following subconjunctival injection of Mf in animals previously hyperimmunized by three subcutaneous injections of Mf. Hyperimmunization may produce an immune response capable of destroying the subconjunctivally injected Mf and preventing their migration into the central cornea.

Ocular immunopathologic findings of experimental onchocerciasis.

Ocular immunopathologic responses of inbred guinea pigs infected with Onchocerca microfilariae from domesticated animals were studied as a laboratory model of human ocular onchocerciasis. A single intracorneal infection of normal guinea pigs with microfilariae produced only minimal ocular lesions. In contrast, intracorneal infection of guinea pigs previously immunized by systemic infection with microfilariae produced intense corneal and uveal inflammation. Transfer of splenic lymphocytes from immunized donors to syngeneic normal recipients substituted effectively for the active immunization. Cell recipients produced marked corneal inflammatory reactions when challenged by a single intracorneal infection. Fresh and cryopreserved microfilariae produced identical reactions. The corneal inflammatory infiltrates were composed primarily of eosinophils, neutrophils, and plasma cells and resembled human onchocercal keratitis. Diethylcarbamazine citrate administration after a challenge intracorneal infection increased the severity of the corneal inflammatory response in immunized animals.